I attempted to transfer a small population of human tumor primary cells into a new well and noticed that there were few signs of adhesion after several hours and ultimately all of the cells had died after O/N incubation. The cells had been growing in culture for about 5 days and had not been passaged since the first plating following digestion of the tumor. While some showed signs of senescence, many looked quite active (image attached).
I had only used 50ul trypsin, in the 24-well plate, for ~1-2 minutes at room temperature and quenched with 1.5ml media with 10% FBS. Could it be that fresh human primary cells are particularly sensitive to trypsinization? Would the cells benefit from a wash step following trypsinization? Are other non-enzymatic methods of dissociation preferred for early primary cells?
you could try a solution of 2 mM EDTA in PBS. It does not digest anything and still inactivates several adhesive proteins on the surface of the cells. The accutase is milder, but I have not tested with primary cells. I just wanted to clear a concept: Although many proteins on the surface are digested by trypsin, integrin beta 1 is not. By incubating cells with high concentrations of trypsin for 20 minutes, the "Fibronectin Receptor" was found: Integin beta 1 alpha 5.
The other posibility is that the tumor cells are not able to produce fast enough extracellular matrix proteins. You could try to give the a little bt of help, by coating the dish previously with collagen of fibronectin... once the touch grownd, they should start the cell cycle without any big problem. Good luck
It sounds like you did everything correctly. I do know that TRAMP tumour cells are particularly sensitive--including to trypsin--so it would not surprise me that human tumour cells might also be. At the very least, pellet the cells with gentle centrifugation (whatever the minimum RCF is for your cells), discard the supernatant, and resuspend them in fresh media.
To my mind, and according previous experience, as trypsin way of acting (protease) is to destroy membrane adhesive proteins, which allow cell to adhere, the simple use of trypsin is more "stressing" for cells than gentle scratching with a litte plastic stick that less injure cells.
Now, why primary culture could be more sensitive to trypsin (I had also see this) ? Perhaps because that at this step of culture, adhesive proteins expressed are less important (in number) on the cell surface, and also, phenotype expression of the cell different: as selectins, whose adhesion properties are less important than integrins (see mechanism of rolling, then strong adhesion of monocyte on endothelium on arterial vessel wall during extravasation of these cells).
Best regards.
Didier J
Thank you for the response, I will give washing a try the next chance I get. I am also planning on scaling back from 0.25% to 0.05% trypsin. Someone also suggested that the primary cells may have a dependence on autocrine signaling and could possibly benefit from using a bit of the "conditioned" media that can be saved prior to passage.
Didier, yes. It would make sense that the primary cells strongly rely on adhesion signals for survival (more so than immortalized cells), but what is odd is that these cells were sitting in digestion solution for several hours prior to the initial plating - they seemed to tolerate that, but not the trypsinization.
I suppose it is also possible that the cells, in spite of appearances, had become senescent and therefore did not re-adhere.
Best,
PVB
Hi, I am at home since it is weekend. I will try to send references on this first hour monday. Yes, primary cells are more sensitive to tripsine. That is why researchers use a physical method to harvest primary cells from culture.
Hello, yes primary cells are very sensitive to trypsin. So usually do mechanical dissociation till 10 th passage. Then used trypsin for subculture.
Dear Pierre,
" these cells were sitting in digestion solution for several hours". Yes, you are right; time for tissue digestion before primary culture can be long, but first steps use different enzymes as collagenase ie , to dissociate and digest collagen, reticulin fibers, the intercallular structures, to liberate free cells; and trypsin and chymostrypsin act more specifically on surface membrane proteins.
Best regards
Didier J
Prof John Beard of Edinburgh University wrote in 1904 that the combined action of trypsin AND chymotrypsin killed cancer cells through the digestion of the protective protein of the trophoblast. He also stated that although trypsin in vitro was effective, it would be toxic in vivo to use the amount required to kill cancer cells without the balance of chymotrypsin. Your finding is very interesting and encouraging.
Cells are cultivated in flascons without using a trypsin, flushing the bottom of this glass jars with spray and that is enough for lifting-off the cells.
You can use a mechanical method like scraping instead of trypsin. Also, you could try accutase since some people have reported better outcomes.
"Accutase: is a mixture of proteolytic and collagenolytic enzymes isolated from crustacean; is mammary component free; for some cell types no need for inactivation or removal during passaging; is very gentle for cells, thus most of the surface proteins are intact after passaging; has to be stored at 4°C; Accutase is inactivated automatically after 1 h at 37°C." http://www.cellntec.com/sites/default/files/cellntec_general_cultivation_protocol.pdf
"The advantages of Accutase over the traditional Trypsin/EDTA treatment are that it is less damaging to cells leading to increased viability and carries a lower risk of introducing adventitious agents into cell cultures because it does not contain any mammalian or bacterially derived proteins." http://www.thermo.com.cn/Resources/200802/productPDF_26369.pdf
Here is one example of a protocolo comparing accutase to trypsine on neural stem cells
"Within 4 days after dissociation, a large proportion of the remaining cells in trypsinized rodent NSC cultures died (Fig. 1c). In contrast, dissociation using Accutase not only increased the initial number of viable cells but also significantly improved the survival of cells. As a consequence, an increase in total cell and sphere numbers was measured in Accutase-treated cultures" http://www.nature.com/labinvest/journal/v83/n7/full/3780683a.html
you could try a solution of 2 mM EDTA in PBS. It does not digest anything and still inactivates several adhesive proteins on the surface of the cells. The accutase is milder, but I have not tested with primary cells. I just wanted to clear a concept: Although many proteins on the surface are digested by trypsin, integrin beta 1 is not. By incubating cells with high concentrations of trypsin for 20 minutes, the "Fibronectin Receptor" was found: Integin beta 1 alpha 5.
The other posibility is that the tumor cells are not able to produce fast enough extracellular matrix proteins. You could try to give the a little bt of help, by coating the dish previously with collagen of fibronectin... once the touch grownd, they should start the cell cycle without any big problem. Good luck
Again, thanks all for the feedback...lots of good points and suggestions. I'm intrigued by the particular finding that beta 1 alpha 5 integrin was found to be particular stable in the presence of trypsin.
As a side note, this has been my first foray with ResearchGate and I'm really impressed. Thanks again for all the suggestions!
I think, in next time, trouble shooting, it is always better to wash the cells in the concerned media atleast two times after trypsinization. It would defiitely benefit the procedure.
@KV Narasimhulu: Washing the cells in the medium will only help if there's serum in the medium. For serum-free medium (which is required to grow a number of primary cells), there's no point washing with medium (since serum neutralizes trypsin). In case of such serum-free medium, Pierre might have to use commercially available trypsin neutralization solution (the one from ATCC works great..!!) to mitigate trypsin action.
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